Nanoparticles (NPs) have shown tremendous potential for the delivery of therapeutics and diagnostics to sites of disease in vivo.1-6 Traditionally, research has been focused on the design of NPs capable of passive accumulation within diseased tissue,7-10 or via an active targeting method employing displayed ligands for overexpressed receptors on tumor associated cells.2,3,6,11-13 By contrast, the focus of the Gianneschi lab has been on active targeting methods for accumulating NPs through physical changes in morphology and nanoscale structure in response to enzymatic signals associated with tumor tissue.14-17 In these active nanocarriers, it is their dynamic stimuli-responsive behavior that is the critical element in their functionality. However, very little is known about te fundamental mechanisms that underlie morphology transitions of this kind, primarily due to a lack of adequate techniques to observe nanoscale dynamic systems in real-time.6,18 This lack of understanding greatly limits the development of next-generation active nanocarriers where optimization is dependent on understanding basic mechanisms of action and response in complex liquid milieu. The long-term objective of the proposed research is to develop fundamental knowledge about the nanoscale mechanisms and kinetics of dynamic stimuli-responsive materials and processes. In situ liquid cell transmission electron microscopy (LCTEM) techniques18,19 will be developed and established as unique tools for imaging organic nanostructures in physiological liquids. The proposed work represent the first efforts to utilize TEM for imaging this type of material in liquid, which can lay the foundation for a future n which LCTEM is routinely used to characterize dynamic biological nanostructures including viruses and cellular components. Here, two different stimuli-responsive NP systems will be studied, one enzyme-responsive14-17 and one pH-responsive, both of which undergo morphological transformations after stimuli-induced reactions occur at the liquid-particle interface. Novel ultra-thin graphene liquid cells20,21 will be constructed for LCTEM characterization, overcoming issues related to image contrast and detection22-25 of these hydrated organic structures (Aim 1). Initial LCTEM experiments will image the synthesized and post-reaction morphologies at various stages of reaction progression using pre-mixed solutions, providing the first clues into the dynamic transformation processes involved. Ultimately, liquid-flow capabilities, using enzyme or acidic solution, will be employed with the graphene liquid cells to observe the complete stimuli-responsive behaviors, from the initial reaction events through to their final equilibrium morphologies (Aim 2). The acquired in situ LCTEM videos will be analyzed visually and by multi-target tracking computational methods26-28 to elucidate the mechanisms and kinetics of transformation. The results will expose the limiting and enabling steps for rapid transformation. With this knowledge, synthetic chemists can intelligently modify the chemistry of the micelle building blocks to create new nanocarriers with tailored stimuli-responsive transformation behaviors for improved in vivo behavior.